The objectives of this continuing project are to study the biology and biochemical regulation of a pathway of apoptosis in CD4 T lymphocytes that is mediated by engagement of the Fas death receptor. The importance of this form of apoptosis (called activation-induced cell death [AICD]) in the maintenance of self-tolerance is illustrated by the autoimmune diseases that develop in mice and humans with inherited defects in Fas or Fas ligand (FasL). The specific aims of the project are the following. 1. Biology of Fas-mediated AICD. In this aim we will examine the hypothesis that AICD is induced by repeated T-cell activation, and define the conditions of antigen exposure that trigger this death pathway. We will use homogeneous T-cells from T-cell receptor (TCR) transgenic mice to test if and when AICD is triggered by immunization with antigen in adjuvant, infection with viruses expressing the antigen, and exposure to the antigen expressed as a systemic self protein. We will determine the factors that influence the choice between T-cell proliferation and apoptosis, including the concentration of antigen, the frequency and persistence of antigen exposure, the frequency of responding T-cells, and the amounts of IL-2 produced by the T-cells. We will define the effects of defective AICD on the survival of T-cells, the functional interactions between antigen-specific B- and T-cells, and the development of autoimmunity. We will also examine the consequences of disrupting Fas signals in T-cells by expressing the Fas antagonist, FLIP, or a dominant negative mutant of the adapter protein, FADD, by retrovirus-mediated gene transfer. 2. Regulation of Fas-mediated AICD. In this aim we will define the biochemical correlates of T-cell sensitivity to AICD induced by different types of antigen exposure in vitro and in vivo, focusing on the expression of the pro-apoptotic molecule FasL, the anti-apoptotic molecule FLIP, caspases, and other possible inhibitors of death pathways. We will examine the mechanisms by which IL-2 paradoxically potentiates Fas-mediated death, emphasizing the role of Stat5 signals on the transcriptional activation of FasL and inhibition of FLIP. We will also initiate studies to define the regulation of AICD by other cytokines, specifically IFN-g, and the biochemical mechanisms and functional implications of this regulation. The studies in this project will tell us how an important control mechanism for immune responses and autoreactivity is regulated, and may provide clues about manipulating this pathway therapeutically.